A Membrane Component Essential for Vectorial Translocation of Nascent Proteins across the Endoplasmic Reticulum: Requirements for Its Extraction and Reassociation with the Membrane

Previous reports have shown that rough microsomes treated with high salt (Warren and Dobberstein, 1978, Nature, 273:569-571) or proteases (Walter et al ., 1979, Proc. Nati. Acad. Sci. U. S. A., 76:1,795) are unable to vectorially translocate nascent proteins. Readdition of the high salt or protease extracts restored activity to such inactive rough microsomes. A detailed study was carried out to determine how this factor interacts with the rough microsomal membrane. Proteolytic cleavage was found to be necessary but not sufficient to remove this factor from the membrane. A subsequent treatment with high salt had to be carried out . Endogenous (pancreatic) protease could effect the required cleavage, but low levels of trypsin, clostripain, or elastase were far more efficient. Several proteases were not effective. The minimum level of salt (after proteolysis) required to solubilize the active factor was -200 mM KCI. Salt extracts prepared by treatment with one of the effective proteases were capable of restoring activity to inactive microsomes produced by treatment with one of the others